Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6992-6997, July 1996 Biochemistry

Processing of the leader mRNA plays a major role in the induction of thrS expression following starvation in Bacillus subtilis (mRNA processing/threonyl tRNA synthetase/mRNA stability/antitermination) CIARA&N CONDON, HARALD PUTZER*, AND MARIANNE GRUNBERG-MANAGO Unite Propre de Recherche 9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France Contributed by Marianne Grunberg-Manago, April 1, 1996

ABSTRACT The threonyl-tRNA synthetase gene, thrS, is Changing the specifier codon in the tyrosyl-tRNA synthetase a member of a family of Gram-positive genes that are induced gene, tyrS, from UAC to UUC, switches the specificity of following starvation for the corresponding by a regulation from to (4). Similarly, transcriptional antitermination mechanism involving the cog- changing the UUC codon of the phenylalanyl-tRNA syn- nate uncharged tRNA. Here we show that an additional level thetase gene to ACC permits induction ofpheS by starvation of complexity exists in the control of the thrS gene with the for threonine (8). A second interaction occurs between the mapping of an mRNA processing site just upstream of the NCCA-3' end of the acceptor arm of the uncharged tRNA and transcription terminator in the thrS leader region. The pro- a complementary sequence (UGGN'), which is part of the cessed RNA is significantly more stable than the full-length T-box and is bulged out of the antiterminator structure (4, 5, transcript. Under nonstarvation conditions, or following star- 8). Although the formation of these two interactions is likely vation for an amino acid other than threonine, the full-length to be the critical element in the stabilization of the antitermi- thrS mRNA is more abundant than the processed transcript. nation conformation of the RNA, it is likely that other However, following starvation for threonine, the thrS mRNA tRNA-leader interactions and possibly some factors exists primarily in its cleaved form. This can partly be are required to make this a productive complex in vivo (4, 8). attributed to an increased processing efficiency following We have been studying the mechanism of tRNA-mediated threonine starvation, and partly to a further, nonspecific antitermination in the control of the thrS gene, one of two increase in the stability of the processed transcript under threonyl-tRNA synthetase genes in Bacillus subtilis. Here we starvation conditions. The increased stability ofthe processed show that the thrS leader mRNA is cleaved between the T-box RNA contributes significantly to the levels of functional RNA and the leader terminator structure, and that the cleaved observed under threonine starvation conditions, previously transcript is significantly more stable than the full-length RNA, attributed solely to antitermination. Finally, we show that particulary under starvation conditions. Thus, termination/ processing is likely to occur upstream of the terminator in the antitermination is not the only determinant of the level of leader regions of at least four other genes of this family, functional thrS mRNA in the cell. We show that while the suggesting a widespread conservation of this phenomenon in interaction between uncharged tRNA-Thr and the thrS leader their control. is not necessary for processing to occur, the cleavage efficiency is nonetheless stimulated by threonine starvation. Finally, we Unlike in Escherichia coli, where the genes encoding the show that cleavage between the T-box and the terminator aminoacyl-tRNA synthetases and the amino acid biosynthetic probably occurs in several other members of this family of operons are induced by a wide variety of mechanisms in genes, suggesting that this phenomenon may have a general response to starvation for their cognate amino acid (1, 2), importance in their regulation. many of these genes appear to be regulated by a common transcription antitermination mechanism in Bacillus subtilis and other Gram-positive bacteria (3-6). The genes of this MATERIALS AND METHODS family all have a highly structured leader region of about 300 Bacterial Strains and Culture Conditions. The B. subtilis nucleotides (nt), followed by a Rho-independent transcription strain used for these studies was 168, or derivatives thereof, terminator, just upstream of the translation initiation site. containing thrS-lacZ transcriptional fusions integrated into Upstream of the leader transcription terminator is a highly the amy locus. Strains were in general grown in M9 minimal conserved sequence of about 14 nt, known as the T-box, and medium (9) at 37°C to an OD600 of about 1.0 before harvesting part of the T-box sequence can base pair with a complemen- for RNA isolation. For starvation experiments, cells were tary sequence in the 5' strand of the terminator stem to form grown to an OD600 of about 0.3 in M9 medium, divided, and an alternative, but much weaker, antiterminator structure (2, then starved for 2 h in the presence of either 600 ,ug of 4). The antitermination conformation of the mRNA is thought threonine hydroxamate per ml (Sigma) or 3 ,ug of to be stabilized in each case by the cognate uncharged tRNA, hydroxamate per ml (Sigma), each of which caused about a thus accounting for the induction of these genes by starvation two-fold decrease in growth rate. for their corresponding amino acid (4, 7, 8). Plasmid Construction. The wild-type thrS-lacZ fusion plas- The identity of the stabilizing tRNA is specified by a codon mid (pHMS69) was constructed by cloning a 0.44-kb EcoRI- bulged out of a conserved secondary structure called the BamHI fragment from pHMS25 (8) into the lacZ fusion specifier domain, which occurs early in the leader regions of vector, pHM2 (10), and cut with EcoRI and BamHI. these genes (4, 8). The tRNA is thought to recognize the RNA Isolation. Frozen pellets ofcells (from 20 ml of culture) bulged triplet via a classical codon/anticodon interaction. were resuspended in 4 ml ice-cold TE buffer (pH 8.0). RNA

The publication costs of this article were defrayed in part by page charge Abbreviation: RT, reverse transcriptase. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 6992 Downloaded by guest on September 26, 2021 Biochemistry: Condon et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6993

was isolated by vortex mixing the cells in an ice-cold mixture nuclease mapping experiments on the same RNA preparation. of glass beads, phenol, chloroform, and SDS, as described (3), The Si probe was made by PCR amplification of a fragment followed by two further cold phenol extractions and three containing the promoter and about 850 nt of the thrS gene phenol/chloroform extractions. It was finally precipitated in cloned in pBluescript, using the same labeled oligonucleotide ethanol and resuspended in an appropriate volume of diethyl- as for the RT reactions and the Universal primer as the pyrocarbonate (DEPC)-treated water. upstream oligonucleotide (see Materials and Methods). The Reverse Transcriptase (RT) Assays. RT assays were carried band corresponding to the RT, stop between the T-box and the out as described (10) by using 15 ,ug of total RNA and about terminator was also a product of the Si nuclease reaction (Fig. 1 pmol of 5'-end labeled oligonucleotide. RNA and oligonu- 1B, lane 2), confirming that this 5' end exists in vivo. Control cleotide were heated together at 65°C for 5 min, and then reactions with total E. coli tRNA or the probe alone (lanes 3 frozen in a mixture of dry ice and ethanol and allowed to thaw and 4) did not show a band in this position. Since no such 5' on ice. Reactions contained 2 units of avian myeloblastosis end is observed in in vitro transcription experiments, and the virus RT (Eurogentec, Brussels) and were allowed to run for 160 bp upstream of this site show no promoter activity in vivo 30 min at 48°C. Oligonucleotides were synthesized by Oligo- when fused to lacZ (data not shown), the possibility of Express (Montreuil, France), and the sequences are available transcription initiation from a second gene promoter is elim- on request. inated. We thus concluded that the shorter thrS transcript is a S1 Nuclease Assays. S1 probes were amplified by PCR from cleavage product of the full-length mRNA. plasmids containing either part of the thrS gene or thrS-lacZ The Processed thrS Messenger RNA Is More Stable Than fusions. Probes complementary to the thrS leader and part of the Full-Length Transcript, Particularly Under Starvation the structural gene, which are specific for the native thrS gene Conditions. The processed transcript is likely to have a termi- transcripts, were amplified from pHMS2, a pBluescript- nator structure at both its 5' and its 3' end. Since stable derived plasmid described previously (see figure 3 in ref. 11). secondary structures at the ends of messenger RNAs have Thirty picomol of a 5'-end labeled oligonucleotide (28-mer) been associated with increased stability in E. coli (12-15), we starting at position +503 of the thrS transcript and 30 pmol of examined the stability of the thrS full-length and processed the Universal primer were used in the PCR. Probes specific for mRNAs under normal and starvation conditions. RNA was the wild-type and mutant thrS-lacZ fusions were amplified isolated from B. subtilis strains at 2-min intervals following the from their respective plasmids pHMS69 (wild type; this paper), addition of rifampicin, which prevents further transcription pHMS34 (stop codon mutant), and pHMS29 (deletion of initiation, and subjected to S1 nuclease analysis as above (Fig. specifier domain) described previously (8), and pHMS21 24). The same results were obtained with the RT assay or by (truncated leader fusion) also described previously (3). Thirty Northern blot analysis (data not shown). On minimal medium, picomol of a labeled oligonucleotide starting at codon 42 of the the processed transcript had a half-life of about 2.0 min (Fig. lacZ gene and 30 pmol of an oligonucleotide specific for the cat 2B; Table 1), about three times that of the full-length mRNA gene, also contained on the plasmid, were used for the PCR. (0.7 min). Upon starvation for threonine, the total amount of The PCR products were purified from agarose gels (Jetsorb; thrS mRNA increased dramatically as expected, and the sta- Bioprobe, Montreuil, France) and counted in a scintillation bility of the processed transcript increased significantly to a counter. Fifty micrograms of RNA and 100,000 Cerenkov half-life of about 3.5 min, while the stability of the full-length counts of probe were precipitated together in ethanol and transcript remained unchanged. To see whether the increase in resuspended in 40 ,ll deionized formamide and 10 ,ul of 5 x stability under starvation conditions was specific for threonine, hybridization buffer (0.2 M Pipes, pH 6.4/2 M NaCl/5 mM we repeated the experiment, this time starving for arginine. EDTA). Samples were denatured at 75°C for 10 min, hybrid- Under arginine starvation conditions, the amount of thrS ized overnight at 48°C, and then diluted in 450 gl ice cold S1 readthrough mRNA decreased by half, consistent with previ- reaction buffer [0.25 M NaCl/30 mM NaOAc, pH 4.6/1 mM ous observations using thrS-lacZ fusions (3), and the half-life ZnSO4/5% glycerol/20 ,ug of denatured herring sperm DNA of the processed thrS transcript increased to 3.1 min, whereas per ml/120 units S1 nuclease (Boehringer Mannheim)]. Sam- the full-length transcript remained relatively unstable (0.7 min; ples were incubated for 30 min at 37°C, ethanol precipitated, Table 1). Thus, at least part of the increased stability of the and run on polyacrylamide gels. Quantification of both RT and processed transcript appears to be a nonspecific effect of S1 products was done by using a PhosphorImager apparatus amino acid starvation. (Molecular Dynamics). By comparing the zero-time points (0') for each of the three Half-Life Measurements. For half-life measurements, conditions examined (Fig. 2A), we noticed that the relative RNAs were prepared as described above from cells harvested amount of the processed transcript was significantly higher at 0, 2, 4, and 6 min after addition of rifampicin (Sigma) at 150 following threonine starvation than under either nonstarva- jig/ml. tion or arginine starvation conditions. Starvation for threonine caused a 5-fold increase in the amount of full-length transcript; however, the intensity of the band corresponding to the RESULTS cleaved RNA was increased 35-fold in the same RNA prepa- The thrS Leader Region Is Cleaved Just Upstream of the ration [compare M9 (0') and threonine hydroxamate (THX; Transcription Terminator. Primer extension analysis of total 0') in Fig. 2A and Table 1]. This results in there being 6-fold B. subtilis RNA, using a labeled oligonucleotide complemen- more of the more stable, cleaved form of the thrS mRNA than tary to a sequence about 200 nt downstream of the thrS leader the full-length transcript under inducing conditions, and the transcription terminator, produced three major bands on processed transcript represents 86% of the total thrS mRNA. polyacrylamide gels (Fig. 1B, lane 1). The basic structure of the Under arginine starvation conditions, the intensity of both the thrS gene and RT strategy are shown in Fig. 1A. The band of full-length and the processed RNAs was decreased [compare the lowest molecular weight corresponds to a RT stop at the lanes M9 (0') and arginine hydroxamate (AHX; 0') in Fig. 2A terminator itself, presumably caused by a difficulty in travers- and Table 1], and ratio of processed to full-length mRNAs was ing the rather stable secondary structure, and the largest band 10-fold lower (0.6) than that observed following starvation for corresponds to the full-length messenger RNA generated by threonine and unchanged from that measured on minimal the thrS promoter. We mapped the third RT stop to a point medium. Under noninducing conditions, therefore, the pro- between the T-box and the terminator, 9 nt upstream of the cessed transcript only accounts for 38% of thrS mRNA. terminator stem (Fig. 1C). To determine whether this RT stop Cleavage of the thrS Leader mRNA Can Also Occur Inde- corresponded to a mature 5' end in vivo, we also performed S1 pendently of the Specifier Codon and Specifier Domain. The Downloaded by guest on September 26, 2021 6994 Biochemistry: Condon et al. Proc. Natl. Acad. Sci. USA 93 (1996) A A M9 TIIHX AHX p ,R o' 2' 4' 6 0 2' 4' 6' 0' 2 4' 6' -W t e r m I n a t I o n FL- --p~~~~~~~~~~~~~~~~~~~~~~~~~~~~~S . .. ~~L trmnto readt hrough ......

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0 1 2 3 4 5 6 7 Time after rifampicin (m ins) FIG. 2. (A) S1 analysis showing differential stabilities of the full-length (FL) and processed (scissors) transcripts. RNA (50 ,ug) were used in each lane. The Si probe is marked as P. Lanes marked 0', 2', 4', and 6' correspond to the number of minutes after addition of rifampicin that RNA samples were prepared. Samples harvested on M9 minimal medium (M9), and in the presence of threonine hydrox- amate (THX), and arginine hydroxamate (AHX) are as indicated. (B) Graph of data from the autoradiograph in A, showing the rate of degradation of the individual transcripts.

high levels of induction of thrS expression typically seen under conditions of threonine starvation are thought to be caused by a stabilization of the antiterminator structure by uncharged tRNA-Thr (4, 8). Because the amount of processed RNA was significantly increased under inducing conditions, we wished to see whether the interaction between uncharged tRNA-Thr and the thrS leader were necessary for cleavage to occur. We have previously shown that changing the ACC specifier codon in the thrS leader to a stop codon, UAA, abolishes induction of W

also uninducible by threonine starvation (8). Thus, neither of -259 these mutant leaders is likely to interact with uncharged tRNA-Thr in a productive manner. We used these two mutant fusions to test the necessity of the interaction between the tRNA and the thrS leader for processing. Si analysis of RNA 'L isolated from cells growing in minimal medium showed that - both of these fusions are cleaved with a similar efficiency to a !a- wild-type fusion (Fig. 3, lanes 1-3). Thus, processing can occur :-- W$,.... in the absence of the tRNA-leader mRNA interaction. Be- cause the same amount of RNA (50 ,ug) was used in each lane, :.i,t,$:9S$" and the probes had the same specific activity, the different intensities of the bands in each lane accurately reflect the amount of readthrough transcript for each of these fusions and ''1'#'-'$-'-'1i'

::: correspond quite well to their relative f-galactosidase activi- iS:: ties (see ref. 8). Following threonine starvation, only the wild-type fusion showed an accumulation of readthrough RNA, confirming that neither the specifier codon mutant nor the specifier domain deletion are inducible by uncharged "'M if tRNA-Thr (Fig. 3, lanes 5-7). The processing efficiency of the -136 wild-type fusion differs somewhat from the native thrS gene under both noninducing and inducing conditions for reasons we do not yet understand. Thus, we could not use the mutant fusions to accurately quantify the role played by uncharged tRNA. Leader Is in FIG. 3. Processing in wild-type and various mutant thrS-lacZ Processing Eliminated a Truncated thrS Lead- fusions. Fifty micrograms of RNA was used in each lane. S1 analysis er-lacZ Fusion. We also tested processing efficiency in a of RNA from the wild-type fusion is shown in lanes 1 and 5; the stop thrS-lacZ fusion where the leader region was truncated at an codon mutant in lanes 2 and 6; the deletion of the specifier domain in EcoNI site in the loop of the terminator. Thus, this fusion lacks lanes 3 and 7; and the fusion truncated in the loop of the leader the 3' half of the terminator stem and about 100 nt of terminator in lanes 4 and 8. Sizes are as indicated on the right-hand downstream thrS sequence, but contains all of the sequences side of the gel. Samples harvested on M9 minimal medium (M9) and necessary for the formation of the antiterminator structure. in the presence of threonine hydroxamate (THX) are as indicated. Since cleavage seemed likely to occur in the antitermination Full-length (FL) and processed (scissors) transcripts are as marked. conformation of the leader transcript (see Discussion), we The arrowheads within the gel depict where the processed RNA in the expected the processing efficiency to increase significantly in truncated fusion should migrate. this mutant. However, no trace of the leader processing was thetic operons in B. subtilis. By com- observed under either non-starvation or threonine starvation using oligonucleotides conditions (Fig. 3, lane 4 and 8). All of the radioactivity from plementary to the published sequences of several of these the wild-type induced fusion accumulated in the full-length genes, we performed primer extension experiments on total B. transcript in this deletion mutant (Fig. 3, compare lanes 5 and subtilis RNA to see whether there were RT stops which would 8), suggesting that cleavage of the transcript is abolished, correspond to processing between the T-box and the termi- rather than normal cleavage and decreased stability of the nator structure of their leader regions. Fig. 4A shows the band processed mRNA. Thus, the leader terminator structure, or corresponding to the RT stop in this position in thrZ, and that sequences 3' to it, are necessary for cleavage of the thrS a similar band occurs in three other systems we tested: tyrS, mRNA. serS, and trpS. A similar analysis of the pheS, leuS, and valS Cleavage of the Leader mRNA in the Antiterminator Struc- genes failed to produce conclusive results (data not shown). ture Appears to be Widely Conserved. The second threonyl- However, we noticed a band corresponding to an equivalent tRNA synthetase gene in B. subtilis, thrZ, is normally not position in the ilv-leu operon in a similar experiment by expressed except under threonine starvation conditions or Grandoni et al. (6). Thus, it would appear that cleavage of the when thrS expression is reduced (3, 11). By using RNA isolated mRNA in the antiterminator structure is widely conserved under these conditions, we had previously seen evidence that within this family of genes. There are often one or two minor thrZ was also processed between the T-box and the terminator, bands associated with the RT stop corresponding to the 7 nt upstream of the terminator stem. This suggested that processed RNA, suggesting that the cleavage reaction is not leader processing might play a general role in the regulation of very precise. The proposed major cleavage site of each gene is the aminoacyl-tRNA synthetase genes and amino acid biosyn- estimated in Fig. 4B and was deduced either by RNA sequenc- Downloaded by guest on September 26, 2021 6996 Biochemistry: Condon et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 4. (A) Processing in the genes for various aminoacyl-tRNA synthetases. RT products are shown using oligonucleotide primers specific for the threonyl- (thrS and thrZ), tyrosyl- (tyrS), seryl- (serS), and tryptophanyl- (trpS) tRNA synthetase genes. Sizes are as indicated on the right-hand side of the gel. The full-length transcript (FL), the proposed processing sites (scissors), and the nonspecific RT stop at the terminator structure (T) of each of these genes is marked. The RT enzyme is not processive enough to see the full-length thrZ transcript at >800 nt from the oligonucleotide. (B) Proposed processing sites (scissors) in each of the synthetase genes in A and the ilv-leu biosynthetic operon from ref. 6. The core sequence of the T-box is framed. ing or by sequencing of DNA fragments containing the gene transcript contribute significantly to both basal and induced in question using the oligonucleotide used for the RT reaction levels of thrS expression, since the yield of protein per mRNA as primer (data not shown). is increased. The stability of the processed thrS transcript also increased in response to arginine starvation, although not DISCUSSION quite to the same extent as that provoked by a lack of threonine. This suggests that the increased mRNA half-life We have shown that the thrS leader region is processed at a site seen under starvation conditions may be at least partially due between the T-box and the Rho-independent transcription to a non-specific effect of translation inhibition. Arginine terminator, 9 nt upstream from the terminator stem. This starvation does not lead to an increase in thrS expression would place the cleavage in the loop of the antiterminator because so little of the mRNA is processed, and the increase structure if cleavage were to occur in the antitermination in stability of the cleaved transcript is essentially canceled out conformation (see Fig. 1C). Cleavage of the transcript at this by a decrease in total readthrough. position and subsequent refolding of the terminator structure The intensity of band corresponding to the processed RNA would leave the mature thrS mRNA possessing a "terminator" increases 35-fold (compared with 5-fold for the full-length at both its 5' and its 3' end. In E. coli, stable secondary transcript), and accounts for 86% of the thrS readthrough structures at either end of the transcript have been associated RNA under threonine starvation conditions. Under nonstar- with increased mRNA stability (12-15). However, 5' second- vation conditions, or following starvation for arginine, the ary structures no longer have a stabilizing effect if there are situation is the reverse, and the majority of the thrS mRNA more than four unpaired nucleotides at the beginning of the exists in its more labile, full-length form. Thus, there is a message. Although the analogy with E. coli prompted us to do substantial amplification of the amount of cleaved relative to the initial mRNA stability studies, subsequent experiments the amount of full-length RNA under inducing conditions. (see below) suggest the terminator may play a more funda- While the increase in stability of the processed transcript mental role in the processing event itself. (1.6-fold) accounts for part of its amplification under threo- The processed thrS transcript is about three times as stable nine starvation conditions, the greatest proportion is due to an as the full-length mRNA under normal growth conditions on increased processing efficiency of the full-length RNA. The minimal medium, and becomes as much as 5-fold more stable increased efficiency of thrS mRNA cleavage following threo- than the full-length transcript following starvation for threo- nine starvation would suggest that cleavage occurs in the nine. Thus, processing and the subsequent stabilization of the antitermination conformation of the leader mRNA and could Downloaded by guest on September 26, 2021 Biochemistry: Condon et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6997 be explained as follows. Under nonstarvation conditions, there region were required for proper folding of the mRNA into the is very little transcriptional readthrough and the majority of antitermination conformation for processing. transcripts that escape termination might be predicted to Our primer extension analysis of the genes for other ami- quickly refold into the terminator conformation in the absence noacyl-tRNA synthetases suggests that processing ofthe leader of uncharged tRNA. These full-length transcripts are conse- regions of these genes may be widely conserved. In five of the quently unlikely to be cleaved and are labile. In contrast, under eight B. subtilis synthetase genes we examined, there was a inducing conditions, the vast majority of the full-length tran- clear RT stop that could be mapped to just upstream of the scripts would be predicted to be held in the antitermination terminator structure in each case. There also appears to be an conformation by the uncharged tRNA and subject to cleavage RT stop in an equivalent position in the ilv-leu operon. It and stabilization. The increased cleavage efficiency and sta- obviously remains to be proven that this RT stop corresponds bilization of the processed transcript contribute greatly to the to a mature 5' end in each case; however, its conservation of level of functional thrS mRNA in the cell under inducing position is highly suggestive that these mRNAs are also cleaved conditions. Previously, the increase in expression of genes of in vivo. There is no obvious sequence or positional homology this family following starvation for the cognate amino acid has between the proposed cleavage sites of the various genes. The been solely attributed to antitermination. processing site is not consistently the same distance from either Since uncharged tRNA-Thr is thought to stabilize the the T-box or the terminator stem, the most obvious points of antiterminator structure, and given the proximity of the cleav- reference given their conservation. If the antitermination age site to the proposed site of tRNA-antiterminator interac- conformation of these mRNAs were preferred, as we suspect tion, we decided to test mutations in the thrS leader that would is the case for thrS, cleavage would occur in single stranded allow us to determine whether interaction with the tRNA were regions in the antiterminator structure of thrS, ilv-leu, and serS necessary for processing to occur. We did this experiment in but would occur in a region predicted to be double stranded in the context ofthrS-lacZ fusions we had previously constructed thrZ, tyrS, and trpS. The specificity of the endonuclease activity in the laboratory. Two mutant thrS fusions, the first where the will be the focus of future studies. ACC specifier codon was changed to the UAA stop codon, and the second where the 5' half of the specifier domain was We wish to thank J. Leautey for technical assistance and J. Plum- deleted, both of which would be predicted to prevent proper bridge, J. Ross, and M. Uzan for useful discussion and advice. This interaction with tRNA-Thr, were still processed efficiently work was supported by funds from the Centre National de la Recher- che Scientifique (UPR 9073), Ministere de l'enseignement superieur under nonstarvation conditions in vivo. Thus, processing of the et de la recherche (Contract 92C0315), Universite Paris VII (Contract thrS leader can occur in the absence of the specifier codon or DRED), and from Human Capital and Mobility (ERBCHBICT- specifier domain, and probably, therefore, does not require the 930796) and Federation of European Biochemical Societes to C.C. presence of the uncharged tRNA. However, that cleavage of thrS mRNA is increased specifically under threonine starva- 1. Grunberg-Manago, M. (1987) in Escherichia Coli and Salmonella tion conditions would suggest that, nevertheless, stabilization Typhimurium, Cellular and Molecular Biology, ed. Neidhardt, F. of the antiterminator structure by uncharged tRNA stimulates C. (Am. Soc. for Microbiol., Washington, DC), Vol. 2, pp. processing. 1386-1409. We have previously shown that antitermination can occur 2. Putzer, H., Grunberg-Manago, M. & Springer, M. (1995) in independently of uncharged tRNA-Thr, and that the tRNA- tRNA: Structure, Biosynthesis, and Function, eds. Soll, D. & RajBhandary (Am. Soc. for Microbiol., Washington, DC), pp. independent antitermination mechanism is used to control 293-333. expression of thrS by growth rate (8). Since processing can 3. Putzer, H., Gendron, N. & Grunberg-Manago, M. (1992) EMBO occur in the absence of uncharged tRNA-Thr, but appears to J. 11, 3117-3127. be stimulated by its presence, the data presented in this paper 4. Grundy, F. J. & Henkin, T. M. (1993) Cell 74, 475-482. suggest that processing of the thrS leader is important for the 5. Henkin, T. M. (1994) Mol. Microbiol. 13, 381-387. stabilization of readthrough transcripts resulting from both the 6. Grandoni, J. A., Zahler, S. A. & Calvo, J. M. (1992) J. Bacteriol. tRNA-independent and tRNA-dependent antitermination 174, 3212-3219. mechanisms. 7. Garrity, D. B. & Zahler, S. A. (1994) Genetics 137, 627-636. 8. Putzer, H., Laalami, S., Brakhage, A. A., Condon, C. & Grun- Processing was abolished in the thrS-lacZ fusion truncated berg-Manago, M. (1995) Mol. Microbiol. 16, 709-718. in the loop of the leader terminator, suggesting that the 9. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold terminator itself, or sequences 3' to it, are necessary for Spring Harbor Lab. Press, Plainview, NY). cleavage in vivo. An alternative possibility was that processing 10. Gendron, N., Putzer, H. & Grunberg-Manago, M. (1994) J. occurred as normal in this mutant fusion, but that because the Bacteriol. 176, 486-494. processed RNA lacked the terminator structure at its 5' end, 11. Putzer, H., Brakhage, A. A. & Grunberg-Manago, M. (1990) J. it became unstable to the point of nondetection. However, the Bacteriol. 172, 4593-4602. accumulation of the readthrough mRNA in the full-length 12. Belasco, J. G., Nilsson, A., von Gabain, A. & Cohen, S. G. (1986) form in the mutant, compared with the wild-type fusion under Cell 46, 245-251. 13. Mott, J. E., Galloway, J. L. & Platt, T. (1985) EMBO J. 4, inducing conditions, leads us to favor the former interpreta- 1887-1891. tion. While a requirement for an intact terminator for pro- 14. Chen, C.-Y. A., Beatty, J. T., Cohen, S. N. & Belasco, J. G. (1988) cessing may seem at odds with the idea that cleavage occurs in Cell 52, 609-619. the antitermination conformation, the apparent paradox could 15. Emory, S. A., Bouvet, P. & Belasco, J. G. (1992) Genes Dev. 6, be explained if a pause of RNA polymerase in the terminator 135-148. Downloaded by guest on September 26, 2021